Feb 26, 2020
CODEX Preparation of Reporter 96-Well Plates
- 1University of Florida
- Human BioMolecular Atlas Program (HuBMAP) Method Development CommunityTech. support email: Jeff.spraggins@vanderbilt.edu
Protocol Citation: Jerelyn Nick, Marda Jorgensen 2020. CODEX Preparation of Reporter 96-Well Plates. protocols.io https://dx.doi.org/10.17504/protocols.io.bc2riyd6
Manuscript citation:
Akoya BiosciencesInc,CODEX User Maual REV A.0 2019
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 26, 2020
Last Modified: February 26, 2020
Protocol Integer ID: 33585
Keywords: CODEX, Barcoded Reporter, 96-well plate, reporter mix, nuclear stain, fluorescent dye, oligonucleotide,
Abstract
A CODEX® Reporter is a fluorescent dye conjugated to an oligonucleotide that is complementary to a specific oligonucleotide barcoded antibody. This protocol describes how to prepare 96-well plates of Reporter Mix for use in a CODEX® run. Each well contains Reporters are grouped together in mixtures of three per cycle plus the nuclear stain. During each cycle the Codex instrument dispenses one well on the tissue sample and images the 3 markers and the nuclear stain. At the end of the cycle the Reporters are removed from the tissue by a gentle wash.
Attachments
Guidelines
Plan the CODEX experiment carefully to ensure that each CODEX/barcoded antibody has a unique barcode not shared with any other antibody in the entire multicycle experiment. Assign each antibody a cycle number ensuring that there is only one reporter labled with a given fluorescence/dye in each cycle.
Each cycle can have one, two or three reporters.
Blank cycles, (no reporter) containing nuclear stain only, should be included at the beginning and end of the experiment. (i.e. cycle 1 and last cycle)
Use the attached cycle template to plan the experiment and enter information into the COEX run software.
Use only the CODEX supplied 96-well Plates and Foil seals. Other seals may stick to the instrument during sample loading steps.
Materials
CODEX® KIT
96-well Plates Akoya CODEX 232527
96-well Plate Foil Seals Akoya CODEX 232528
10X CODEX Buffer Akoya CODEX 232119
Assay Reagent Akoya CODEX232120
Nuclear Stain AkoyaCODEX 232121
Reporters (appropriate for barcodes antibodies)
Other Materials
Nuclease-Free Water (not DEPC Treated) Ambion AM9938
Amber colored 1.5 ml tubes USA Scientific Sealrite 1615-5507
Ice bucket
Before start
Thaw Assay reagent, Reporters, and Nulcear Stain and place on ice.
Label a 1.5ml amber microcentrifuge tube for each cycle of the CODEX experiment. Include a Blank cycle at the beginning and end of the experiment.
Prepare the Reporter Stock Solution (Based on the number of cycles and coverslips to be run ):
| Prepare Reporter Stock Solution | ||||||||||
| Number of cycles (wells) | 5 | 9 | 10 | 15 | 18 | 20 | 27 | 36 | 45 | |
| cycles (wells) | ||||||||||
| Nuclease Free Water | 1220 | 2196 | 2440 | 3660 | 4392 | 4880 | 6588 | 8784 | 10980 | |
| 10X CODEX buffer | 150 | 270 | 300 | 450 | 540 | 600 | 810 | 1080 | 1350 | |
| Assay Reagent | 125 | 225 | 250 | 375 | 450 | 500 | 675 | 900 | 1125 | |
| Nuclear Stain | 5 | 9 | 10 | 15 | 18 | 20 | 27 | 36 | 45 | |
| Total | 1500 | 2700 | 3000 | 4500 | 5400 | 6000 | 8100 | 10800 | 13500 | |
| Example: # Coverslips@ 9 cycles | 1 coverslip | 2 coverslips | 3 coverslips | 4 coverslips | 5 coverslips |
Amount of Reporter Stock Solution by Number of Cycles Volumes are in ul.
Prepare the Reporter Master Mix Aliquots for Each Cycle
Label an amber microcentrifuge tube for each cycle. Include tubes for the blank cycles at the beginning and end of the experiment.
Aliquot Reporter Stock Solution into each tube following the chart below (volumes will vary depending on the number of reporters used in an individual cycle (see CODEX Cycle Template).
| Reporter Solutions: Label a 1.5 ml amber tube for each cycle | |||||
| 3 Reporters | 2 Reporters | 1 Reporter | (Blank) 0 Reporters | ||
| 1 Plate (will add 5ul each reporter) | 235 ul | 240 | 245 | 250 | |
| 2 Plates (will add 10 ul each reporter) | 470 ul | 480 | 490 | 500 | |
| 3 Plates (will add 15 ul each reporter) | 705 ul | 720 | 735 | 750 | |
| 4 Plates (will add 20 ul each reporter) | 940 ul | 960 | 980 | 1000 | |
| 5 Plates (will add 25 ul each reporter) | 1175ul | 1200 | 1225 | 1250 |
Reporter Stock Solution volumes by Number of Plates and Number of Reporters per Cycle
Add appropriate Reporters to the Reporter Stock Solution aliquots to make a Master Mix for each cycle
Place reporters on ice.
Briefly, Spin tubes using a bench top centrifuge. Return Reporters to ice.
Pipette Akoya Reporters into the labeled amber tubes containing Reporter Stock Solution. Use volumes listed in step 2.2. column A based on number of reporters in a particular cycle.
Follow the cycle template or the reporter tray template (below).
| CODEX Cycle Template | |||||
| Nuclear Stain | AF 750 | 550 | Cy5 | ||
| cycle 1 Blank cycle | in Reporter Stock | No Reporter | No reporter | No Reporter | |
| cycle 2 (example: 3 antibodies,-750, 550, and Cy5) | in Reporter Stock | Reporter 1 (matches antibody1) | Reporter 2 ( matches antibody2) | Reporter 3 ( matches antibody3) | |
| cycle 3 (example 2 antibodies- 550 and Cy5) | in Reporter Stock | No Antibody in this wavelength | Reporter 4 ( matches antibody4) | Reporter 5 ( matches antibody5) | |
| cycle 4 (example 1 antibody- 550) | in Reporter Stock | No Antibody in this wavelength | Reporter 6 ( matches antibody6) | No Antibody in this wavelength | |
| 5 | |||||
| 6 | |||||
| 7 | |||||
| 8 | |||||
| * | |||||
| last cycle Blank cycle | in Reporter Stock | No Reporter | No reporter | No Reporter |
CODEX Cycle Template.
| Reporter Tray Template | ||||||||
| Cycle 1 | Cycle 2 | Cycle 3 | Cycle 4 | Cycle 5+ | Cycle * | Cycle*+1 | dye/fluor | |
| Blank | Antibody 1 (Reporter 1) | Antibody 4 (Reporter 4) | label any EMPTY channels | Blank | Cy7 | |||
| Antibody 2 (Reporter 2) | Antibody 5 (Reporter 5) | Antibody 7 (Reporter 7) | 550 | |||||
| Antibody 3 (Reporter 3) | Antibody 6 (Reporter 6) | ...etc | Cy5 |
Reporter 96-well Plate Template
Prepare the 96-well Reporter Plate:
Pipette 245 ul of Reporter containing Master Mix into the corresponding cycle well .
Filled 96-well Reporter Plate with foil seal strips in place
Note
Use a different 96-well plate for each Reporter plate prepared. Lightly cover entire rows of wells (i.e. B1-B12) with strips of foil seal.
Keep the filled 96-well plates IN THE DARK to avoid photo bleaching.
Store at 4˚C for up to two weeks.
4 °C